Natural Killer Cells Modified To Express Membrane-Bound Interleukin 15 And Uses Thereof

ABSTRACT

The present invention provides, in certain aspects, a natural killer (NK) cell that expresses all or a functional portion of interleukin-15 (IL-15), and methods for producing such cells. The invention further provides methods of using a natural killer (NK) cell that expresses all or a functional portion of interleukin-15 (IL-15) to treat cancer in a subject or to enhance expansion and/or survival of NK cells.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/550,548, filed Aug. 26, 2019, which is a divisional of U.S.application Ser. No. 15/309,362, filed Nov. 7, 2016, which is the U.S.National Stage of International Application No. PCT/SG2015/050111, filedon May 14, 2015, published in English, which claims the benefit of U.S.Provisional Application No. 61/993,494, filed on May 15, 2014. Theentire teachings of the above applications are incorporated herein byreference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file:

-   -   a) File name: 44591097004_SEQUENCELISTING.txt; created Aug. 5,        2020, 5.26 KB in size.

BACKGROUND OF THE INVENTION

Survival and proliferation of NK cells in vivo requires stimulation bycytokines, such as IL-2 and IL-15. For example, after injection inimmunodeficient mice, activated NK cells became undetectable after 1week but persisted for up to one month if human IL-2 was alsoadministered. Hence, clinical protocols using NK cell infusionstypically rely on IL-2 administration to prolong NK cells survival inpatients. However, IL-2 can have considerable side effects. In additionto fever and chills, IL-2 administration can lead to more serious andpotentially fatal consequences, such as capillary leak syndrome.Decreasing the dose of IL-2 should reduce the risk of side effects butcan result in stimulation of regulatory T cells which can inhibit NKcell function and possibly nullify its anti-cancer effect.

Hence, it would be important to develop alternative ways to promote NKcell expansion and activity in vitro and/or in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingembodiments of the present invention.

FIGS. 1A-1C: Design and expression of IL-15 constructs. 1A. Schematicrepresentation of the wild-type and membrane-bound IL-15 constructs(“wtIL15” and “mbIL15”) used in this study. 1B. Expression of IL-15 onthe surface of NK cells transduced with mbIL15. Expanded NK cells weretransduced with wtIL15, mbIL15 or with a vector containing GFP alone(“Mock”). Flow cytometry dot plots illustrate expression of GFP andIL-15, as detected by an anti-IL15 antibody (R&D Systems) and agoat-anti-mouse secondary antibody conjugated to phycoerythrin (SouthernBiotechnology Associates). Percentage of cells (>98% CD56+CD3-NK cells)in each quadrant is shown. 1C. Secretion of IL-15 by NK cells transducedwith wtIL15. NK cells from 3 different donors were tested in triplicate.Bars indicate mean±SD of ELISA measurements performed in supernatantscollected after 24 and 48 hours of culture without IL-2. No IL-15 wasdetected in the supernatants of mock-transduced cells.

FIGS. 2A-2C: Survival and expansion of NK cells expressing IL-15 invitro. 2A. Percentage of NK cell recovery as compared to input cellsafter 7-day parallel cultures without IL-2 for mock- and mbIL15transduced cells from 15 donors (left panel) and mbIL15- orwtIL15-transduced cells from 9 donors (right panel). Horizontal barsindicate median value. Results of paired t tests are shown. Results ofcultures with IL-2 (10 and 100 IU/mL) are shown in FIG. 6B. Survival andexpansion of mock- and mbIL15-transduced NK cells from 6 donors with lowdose IL-2 (10 IU/mL). 2C. Expansion and long-term survival of NK cellsfrom one donor transduced with mbIL15, wtIL15 or mock-transducedcultured with no IL-2 or low dose IL-2 (results with 100 IU/mL IL2 areshown in FIG. 6). Percentage of NK cell recovery at the indicated daysof culture is shown.

FIG. 3A-3C: Survival and expansion of NK cells expressing mb-IL15 invivo. 3A. Absolute number of human CD45+ cells in peripheral blood ofmice injected with mock- or mbIL15 transduced NK cells with or withoutIL-2 (16 mice total) 7 and 11 days after infusion (P=0.004 with no IL-2,P=0.021 with IL-2 on day 7; P=0.044 and 0.026 on day 11). 3B. Flowcytometric dot plots illustrate the presence of human CD45+, GFP+NKcells in mouse peripheral blood without (top) and with IL-2 treatment(bottom). Percentages of human CD45+ cells with or without GFPexpression is shown. 3C. Percentage of human CD45+ cells in varioustissues of mice injected with mock- or mbIL15 transduced NK cells withor without IL-2 collected 11 days after injection. Collectively,percentages of human CD45+ cells were significantly higher with mbIL15(P<0.001 with no IL-2, P=0.002 with IL-2).

FIGS. 4A-4C: Properties of NK cells expressing mbIL15. 4A. Relativeproportion of GFP+ cells before and after 7 days of culture among NKcell populations transduced with mbIL15 or mock-transduced. Results withNK cells from 13 donors are shown; P<0.001 for mbIL15, not significantfor mock. 4B. Immunophenotypic features of mbIL15-transduced NK cells.Cells marker analysis by flow cytometry was performed on NK cellscultured for 48 hours without IL-2. All results are summarized in theTable. 4C. Mock- and mbIL15-transduced NK cells were cultured for 48hours without IL-2 and cell lysates were analyzed by Kinex AntibodyMicroarray (Kinexus). Of 809 anti-phosphoprotein antibodies tested,shown are those whose signals had a Z-ratio >0.5 and a % Error Range<100. Bars indicate percent signal change in NK cells expressing mbIL15as compared to the normalized intensity in mock-transduced NK cells.

FIGS. 5A-5D: Anti-tumor capacity of NK cells expressing mbIL15. 5A.Results of 24-hour cytotoxicity assays with mbIL15- and mock-transducedNK cells from 9 donors against the Nalm-6, U937, K562, Daudi, SK-BR-3,and ES8 cell lines at 1:4 and 1:1 E:T ratio (15 experiments at eachratio; P<0.001 for both). Results obtained with individual cell lines in4-hour and 24-hour cytotoxicity assays are shown in FIG. 7. 5B. NK cellsexpressing mbIL15 have an increased release of lytic granules in thepresence of target cells. Percentage of CD107a+ NK cells after 4-hourcytotoxicity assays at 1:1 E:T. Results with NK cells from 3 donorsagainst 2 cell lines are shown (P=0.007). 5C. NK cells expressing mbIL15exert anti-tumor activity in vivo. NOD-SCID-IL2RGnull mice were injectedi.p. with 1×10⁴ U937 cells labeled with luciferase. In 3 mice, notreatment was given (“No NK”), while 4 mice received mock-transduced NKcells (1×10⁷ i.p.) on days 3 and 7, and 4 other mice mbIL15-transducedNK cells at the same dose and schedule. Results of in vivo imaging oftumor growth are shown (ventral images). 5D. Overall survivalcomparisons of mice in the different treatment groups. Mice wereeuthanized when bioluminescence reached 1×10¹¹ photons/second. P valuesfor log rank test of the 3 curves, and for comparisons between each of 2curves are shown.

FIGS. 6A-6C: Survival and expansion of NK cells expressing IL-15 invitro. 6A. Expansion of NK cells expressing mbIL15 in the absence ofIL-2 is suppressed by an anti-IL-15 neutralizing antibody. Symbols showmean (±SD; n=3) NK cell recovery during culture as compared to inputcells in experiments with NK cells transduced with mbIL15. 6B.Percentage of NK cell recovery as compared to input cells after 7-dayparallel cultures with low- (10 IU/mL) and high-dose (100 IU/mL) IL-2for mock-, mbIL15- and wtIL15-transduced cells from 6 donors. Horizontalbars indicate median value. Results of paired t tests are shown. 6C.Expansion and long-term survival of NK cells from one donor transducedwith mbIL15 or wtIL15 and cultured with 100 IU/mL IL2. Percentage of NKcell recovery at the indicated days of culture is shown.

FIGS. 7A-7B: Anti-tumor capacity of NK cells expressing mbIL15. Resultsof 4-hour (7A) and 24-hour cytotoxicity assays (7B) with mbIL15- andmock-transduced NK cells against the Nalm-6, U937, K562, Daudi, SK-BR-3,and ES8 cell lines at 1:4, 1:2 and 1:1 E:T ratio are shown. Each symbolindicates mean±SD cytotoxicity in experiments with NK cells from 3different donors for U937, K562, ES8, and 2 donors for Nalm-6, Daudi andSK-BR-3, all performed in triplicate (P<0.001 for all experiments).

FIGS. 8A-8C: Anti-tumor capacity of NK cells expressing mbIL15.NOD-SCID-IL2RGnull mice were injected i.p. with 1×10⁵ ES8 cells labeledwith luciferase. In 7 mice, no treatment was given (“No NK”), while 11mice received mock-transduced NK cells (1×10⁷ i.p.) on day 3, and 12other mice mbIL15-transduced NK cells at the same dose and schedule. 8A.Results of in vivo imaging of tumor growth. Ventral images of the 4 micewith the highest tumor signal in each group are shown. 8B. Results of invivo imaging of tumor growth. Each symbol corresponds to onebioluminescence measurement (photon/second relative day 3 measurementsin each mouse). 8C. Overall survival comparisons of mice in thedifferent treatment groups. Mice were euthanized when bioluminescencereached 1×10¹⁰ photons/second. P values for log rank test of the 3curves, and for comparisons between each of 2 curves are shown.

FIG. 9 shows the nucleotide sequence (SEQ ID NO: 1) and the amino acidsequence (SEQ ID NO: 2) of membrane bound IL-15.

FIG. 10 shows the nucleotide sequence (SEQ ID NO: 3) and amino acidsequence (SEQ ID NO: 4) of human IL-15 (NCBI Reference Sequence:NM_000585.4).

FIGS. 11A-11C: mbIL15 stimulates NK cells by cis presentation. 11A. NK92cells were transduced with mbIL15 (left) or wtIL15 (right) in a vectorcontaining GFP, sorted to obtain 100% GFP+ cells and co-cultured withuntransduced NK92 cells at a 1:1 ratio. Shown is percentage of cellrecovery (±SD; n=3) after culture for GFP+ and GFP− cells, relative tothe number of cells at the beginning of the culture. 11B. NK92 cellsexpressing mbIL15 or untransduced were co-cultured with K562 cells (“K”)either transduced with mbIL15 or untransduced at 1:2 ratio in thecombinations shown. K562 cells were labeled with PKH26 (Sigma) andtreated with Streck cell preservative (Streck, Omaha, Nebr.) to preventcell division before culture. Shown is percentage of NK92 cell recovery(±SD; n=3) after culture, relative to cell numbers at the beginning ofthe culture. 11C. Proliferation of NK92 cells expressing mbIL15 comparedto that of untransduced NK92 cells in the presence of increasingconcentrations of exogenous IL-15. Cultures were performed in theabsence of IL-2 (left), or with IL-2 at 10 IU/mL (center) or 100 IU/mL(right). Shown is percentage of cell recovery (±SD; n=3) after culturerelative to the number of cells at the beginning of the culture.

FIGS. 12A-12C. Expression and function of KIRs in mb15-NK cells. 12A. NKcell subsets defined by their KIR expression before transduction, andafter mock- or mb15-transduction. Flow cytometric dot plots show resultsof staining with anti-KIR antibodies in CD56+CD3− cells from 2 donors.Percentages of KIR+ cells are shown. 12B. Results of CD107a expressionin CD158a-positive and CD158a-negative subsets after 4-hour culture with721.221 cells or the same cells expressing the CD158a-binding Cw6 HLA.Shown are mean (±SD) of 4 independent experiments with NK cells from 3donors (** P<0.0001; *P=0.0002). 12C. Results of IFNγ secretion in thesame experiments shown in 12B (** P<0.0001).

FIGS. 13A and 13B: Antibody-dependent cell cytotoxicity (ADCC) of NKcells expressing mbIL15. Results of 4-hour ADCC assays with mbIL15- andmock-transduced NK cells against (13A) Daudi and (13B) SK-BR-3 in thepresence of Rituximab or Trastuzumab, respectively; IgG at the sameconcentration of the immunotherapeutic antibodies (1 μg/mL) was used asa control. Each symbol indicates mean±SD cytotoxicity in experimentswith NK cells from each donor in triplicate. In the presence ofimmunotherapeutic antibodies, mbIL15-NK cells exerted significantlyhigher ADCC than mock-transduced cells (P<0.001 for either donor intests with Daudi or SK-BR-3). Cytotoxicity by mbIL15-NK cells withoutantibody was also significantly higher (P<0.001 for either donor intests with Daudi or SK-BR-3).

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The well-established anti-leukemic activity of natural killer (NK) cellsindicates therapeutic potential for NK cell infusions. NK cell survivaland, hence, cytotoxicity requires cytokine support. Described herein areexperiments investigating whether expression of interleukin-15 (IL-15)in a non-secretory, membrane-bound form could sustain NK cell growth.The human IL15 gene was linked to that encoding CD8a transmembranedomain (“mbIL15”). After retroviral transduction, human NK cellsexpressed mbIL-15 on the cell surface but IL-15 secretion wasnegligible. Survival and expansion of mbIL15-NK cells without IL-2 wasvastly superior to that of mock-transduced cells (after 7-day culture,P<0.0001, n=15), and to that of NK cells secreting non-membrane boundIL-15 (P=0.025, n=9); viable mbIL15-NK cells were detectable for up to 2months. In immunodeficient mice, mbIL15-NK cells expanded without IL-2,and were detectable in all tissues examined (except brain) in muchhigher numbers than mock-transduced NK cells (P<0.001). Expansion invitro and in vivo further increased with IL-2. The primary mechanism ofmbIL15 stimulation was autocrine; it activated IL-15 signaling andanti-apoptotic signaling. Cytotoxicity against leukemia, lymphoma andsolid tumor cell lines was consistently higher with mbIL15-NK cells.Median 24-hour cytotoxicity at 1:4 E:T was 71% versus 22% withmock-transduced cells; at 1:1 E:T, it was 99% versus 54% (P<0.0001).Increased anti-tumor capacity was also evident in immunodeficient miceengrafted with leukemia (U937) or sarcoma (ES8) cells. Thus, mbIL15conferred independent growth to NK cells and enhanced their anti-tumorcapacity. Infusion of mbIL15-NK cells allows NK cell therapy without theadverse effects of IL-2.

Accordingly, provided herein is a (one or more; a plurality) cell thatexpresses all or a functional portion of interleukin-15 (IL-15), whereinthe cell is a cell that responds to IL-15. A cell that responds to IL-15includes a cell in which one or more of its activities are regulated byIL-15. Examples of such cells include natural killer (NK) cells,T-cells, dendritic cells and moncytes. The one or more (e.g., isolated)cells can express all or a functional portion of IL-15 as amembrane-bound polypeptide, as a secretory protein or as a combinationthereof.

In one aspect, the invention is directed to a natural killer (NK)cell(s) that expresses all or a functional portion of interleukin-15(IL-15). The one or more (e.g., isolated) NK cells can express all or afunctional portion of IL-15 as a membrane-bound polypeptide, as asecretory protein or as a combination thereof.

As used herein, “Natural Killer Cells” (“NK cells”) refer to a type ofcytotoxic lymphocyte of the immune system. NK cells provide rapidresponses to virally infected cells and respond to transformed cells.Typically immune cells detect peptides from pathogens presented by MajorHistocompatibility Complex (MHC) molecules on the surface of infectedcells, triggering cytokine release, causing lysis or apoptosis. NK cellsare unique, however, as they have the ability to recognize stressedcells regardless of whether peptides from pathogens are present on MHCmolecules. They were named “natural killers” because of the initialnotion that they do not require prior activation in order to killtarget. NK cells are large granular lymphocytes (LGL) and are known todifferentiate and mature in the bone marrow from where they then enterinto the circulation.

In some aspects, the NK cell is a mammalian NK cell. Examples of“mammalian” or “mammals” include primates (e.g., human), canines,felines, rodents, porcine, ruminants, and the like. Specific examplesinclude humans, dogs, cats, horses, cows, sheep, goats, rabbits, guineapigs, rats and mice. In a particular aspect, the mammalian NK cell is ahuman NK cell.

As used herein “Interleukin-15” (“IL-15”) refers to a cytokine thatregulates T and NK cell activation and proliferation. This cytokine andinterleukin 2 share many biological activities. They are found to bindcommon receptor subunits, and may compete for the same receptor, andthus negatively regulate each other's activity. The number of CD8+memory cells is shown to be controlled by a balance between IL-15 andIL-2. This cytokine induces the activation of JAK kinases, as well asthe phosphorylation and activation of transcription activators STATS,STATS, and STAT6 and may increase the expression of apoptosis inhibitorBCL2L1/BCL-x(L), possibly through the transcription activation activityof STAT6, and thus prevent apoptosis.

A “functional portion” (“biologically active portion”) of IL-15 refersto a portion of IL-15 that retains one or more functions of full lengthor mature IL-15. Such functions include the promotion of NK cellsurvival, regulation of NK cell and T cell activation and proliferationas well as the support of NK cell development from hematopoietic stemcells.

As will be appreciated by those of skill in the art, the sequence of avariety of IL-15 molecules are known in the art. In one aspect, theIL-15 is a wild type IL-15. In some aspects, the IL-15 is a mammalianIL-15 (e.g., Homo sapiens interleukin 15 (IL15), transcript variant 3,mRNA, NCBI Reference Sequence: NM_000585.4; Canis lupus familiarisinterleukin 15 (IL15), mRNA, NCBI Reference Sequence: NM_001197188.1;Felis catus interleukin 15 (IL15), mRNA, NCBI Reference Sequence:NM_001009207.1). Examples of “mammalian” or “mammals” include primates(e.g., human), canines, felines, rodents, porcine, ruminants, and thelike. Specific examples include humans, dogs, cats, horses, cows, sheep,goats, rabbits, guinea pigs, rats and mice. In a particular aspect, themammalian IL-15 is a human IL-15.

All or a functional portion of IL-15 can be expressed by one or more NKcells (as a membrane-bound and/or secreted polypeptide) in a variety ofways. For example, all or a functional portion of the IL-15 can beexpressed within the NK cell and secreted from the NK cell and/or can belinked (conjugated; fused) directly or indirectly (e.g., ionic,non-ionic, covalent linkage) to the surface (e.g., at the surface, orwithin the membrane, of an NK cell) of the NK cell using any of avariety of linkers known in the art (Hermanson, G., BioconjugateTechniques, Academic Press 1996). In particular aspects, all or afunctional portion of the IL-15 is linked to all or a portion of atransmembrane protein. In one aspect, the NK cell expresses a fusionprotein comprising all or a portion of IL-15 fused to all or a portionof a transmembrane protein. In a particular aspect, the portion of thetransmembrane protein comprises all or a portion of a transmembranedomain of the transmembrane protein.

As used herein, a “transmembrane protein” or “membrane protein” is aprotein located at and/or within a membrane such as the phospholipidbilayer of a biological membrane (e.g., biomembranes such as themembrane of a cell). Membrane proteins enable the membrane to carry outits distinctive activities. The complement of proteins attached to amembrane varies depending on cell type and subcellular location. Someproteins are bound only to the membrane surface, whereas others have oneor more regions buried within the membrane and/or domains on one or bothsides of the membrane. Protein domains on the extracellular membranesurface are generally involved in cell-cell signaling or interactions.Domains lying along the cytosolic face of the membrane have a wide rangeof functions, from anchoring cytoskeletal proteins to the membrane totriggering intracellular signaling pathways. Domains within themembrane, referred to herein as “transmembrane domains”, particularlythose that form channels and pores, move molecules across the membrane.A “transmembrane domain”, is a three-dimensional protein structure whichis thermodynamically stable in a membrane (e.g., a membrane of a vesiclesuch as a cell). Examples of transmembrane domains include a singlealpha helix, a stable complex of several transmembrane alpha helices, atransmembrane beta barrel, a beta-helix of gramicidin A, or any otherstructure. Transmembrane helices are usually about 20 amino acids inlength.

Typically, membrane proteins are classified into two broadcategories—integral (intrinsic) and peripheral (extrinsic)—based on thenature of the membrane-protein interactions. Most biomembranes containboth types of membrane proteins.

Integral membrane proteins, also called intrinsic proteins, have one ormore segments that are embedded in the phospholipid bilayer. Integralmembrane proteins include transmembrane proteins and lipid-anchoredproteins. Most integral proteins contain residues with hydrophobic sidechains that interact with fatty acyl groups of the membranephospholipids, thus anchoring the protein to the membrane. Most integralproteins span the entire phospholipid bilayer. These transmembraneproteins contain one or more membrane-spanning domains as well asdomains, from four to several hundred residues long, extending into theaqueous medium on each side of the bilayer. Typically, themembrane-spanning domains are one or more (e.g., about 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more) a helices and/or β strands. Membrane-spanningα-helical domains are typically embedded in membranes by hydrophobicinteractions with the lipid interior of the bilayer and probably also byionic interactions with the polar head groups of the phospholipids(e.g., glycophorin). The structure of β strands are typically in theform of membrane spanning barrels (e.g., porin). Some integral proteinsare anchored to one of the membrane leaflets by covalently bound fattyacids. In these proteins, the bound fatty acid is embedded in themembrane, but the polypeptide chain does not enter the phospholipidbilayer. Some cell-surface proteins are anchored to the exoplasmic faceof the plasma membrane by a complex glycosylated phospholipid that islinked to the C-terminus (e.g., glycosylphosphatidylinositol, alkalinephosphatase). Some cytosolic proteins are anchored to the cytosolic faceof membranes by a hydrocarbon moiety covalently attached to a cysteinenear the C-terminus (e.g., prenyl, farnesyl, and geranylgeranyl groups).In another group of lipid-anchored cytosolic proteins, a fatty acylgroup (e.g., myristate or palmitate) is linked by an amide bond to theN-terminal glycine residue.

Peripheral membrane proteins, or extrinsic proteins, do not interactwith the hydrophobic core of the phospholipid bilayer. Instead they areusually bound to the membrane indirectly by interactions with integralmembrane proteins or directly by interactions with lipid polar headgroups. Peripheral proteins localized to the cytosolic face of theplasma membrane include the cytoskeletal proteins spectrin and actin inerythrocytes and the enzyme protein kinase C. This enzyme shuttlesbetween the cytosol and the cytosolic face of the plasma membrane andplays a role in signal transduction. Other peripheral proteins,including certain proteins of the extracellular matrix, are localized tothe outer (exoplasmic) surface of the plasma membrane.

Examples of transmembrane proteins include a receptor, a ligand, animmunoglobulin, a glycophorin or a combination thereof. Specificexamples of transmembrane proteins include CD8α, CD4, CD3ε, CD3γ, CD3δ,CD3ζ, CD28, CD137, FcεRIγ, a T-cell receptor (TCR such as TCRα and/orTCRβ), a nicotinic acetylcholine receptor, a GABA receptor, or acombination thereof. Specific examples of immunoglobulins include IgG,IgA, IgM, IgE, IgD or a combination thereof. Specific examples ofglycophorin include glycophorin A, glycophorin D or a combinationthereof.

In addition to being linked to all or a portion of a transmembraneprotein, all or a functional portion of the IL-15 can be linked to othercomponents such as a signal peptide (e.g., a CD8α signal sequence), aleader sequence, a secretory signal, a label (e.g., a reporter gene),etc. In a particular aspect, the all or a functional portion of IL-15 isfused to a signal peptide of CD8α and all or a portion of atransmembrane domain of CD8α.

In another aspect, the invention is directed to a method of producing anatural killer (NK) cell that expresses all or a functional portion ofinterleukin-15 (IL-15). All or a portion of the IL-15 can be expressedas a membrane-bound polypeptide, a secreted polypeptide or as acombination thereof. The method comprises introducing nucleic acidencoding all or a functional portion of IL-15 into the one or more NKcells. In one aspect, the nucleic acid encoding all or a functionalportion of IL-15 is linked (e.g., fused) to all or a portion of atransmembrane protein. Alternatively, or in addition, nucleic acidencoding all or a functional portion of IL-15 is introduced into the NKcell (e.g., wild type IL-15). As will be apparent to those of skill inthe art, aspects in which nucleic acid encoding all or a functionalportion if IL-15 and all or a functional portion of IL-15 fused to allor a portion of a transmembrane protein is introduced in to NK cell, canbe done so using a single nucleic acid or multiple (e.g., separate; two)nucleic acids. The NK cell is maintained under conditions in which allor a functional portion of the IL-15 is expressed as a membrane-boundpolypeptide and/or as a secreted polypeptide thereby producing a NK cellthat expresses all or a functional portion of IL-15 as a membrane-boundpolypeptide and/or as a secreted polypeptide. In a particular aspect,nucleic acid encoding all or a functional portion of IL-15 is fused to asignal peptide of CD8α and all or a portion of a transmembrane domain ofCD8α is introduced into the NK cell.

In yet another aspect, the invention is directed to a method ofenhancing expansion and/or survival of NK cells (e.g., in vitro, exvivo, and/or in vivo). The method comprises introducing nucleic acidencoding all or a functional portion of IL-15. Nucleic acid encoding allor a portion of the IL-15 (e.g., wild type IL-15) and/or encoding all ora functional portion of IL-15 fused to all or a portion of atransmembrane protein can be introduced into the NK cell. Thus, the NKcell can express all or a functional portion of IL-15 as amembrane-bound polypeptide, a secreted polypeptide or as a combinationthereof. The NK cells are maintained under conditions in which all or aportion of the IL-15 is expressed as a membrane-bound polypeptide, asecreted polypeptide or as a combination thereof and in which the NKcells proliferate. In a particular aspect, nucleic acid encoding all ora functional portion of IL-15 is fused to a signal peptide of CD8α andall or a portion of a transmembrane domain of CD8α is introduced intothe NK cell. In some aspects, the method can further comprise contactingthe NK cells comprising membrane-bound IL-15 and/or secreted IL-15 withIL-2. In some aspects, the concentration of IL-2 is from about 10 IU/mlto about 1000 IU/ml. In other aspects, the concentration of IL-2 isabout 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280,300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560,580, 600, 620, 640, 660, 680, 700, 720 740, 760, 780, 800, 820, 840,860, 880, 900, 920, 940, 960, 980 IU/ml.

As will be apparent to those of skill in the art, a variety of methodsfor introducing nucleic acid (e.g., transfection, transduction, and/ortransposon system) encoding all or a functional portion of IL-15 as atransmembrane polypeptide and/or as a secreted polypeptide into a NKcell can be used. Examples of such methods include chemical-basedmethods (e.g., involving the use of calcium phosphate; highly branchedorganic compounds (e.g., dendrimers); liposomes (lipofection); and/orcationic polymers (e.g., DEAE dextran; polyethylenimine)),non-chemical-based methods (e.g., electroporation; cell squeezing;sonoporation; optical transfection; impalefection; hydrodynamicdelivery), particle-based methods (e.g., gene gun; magnetofection;particle bombardment), vector-based methods (e.g., vectors includingviral vectors such as retroviral vector, lentiviral vectors, adenoviralvectors, etc.), nucleotransfection, transposon-based methods (e.g.,Sleeping Beauty, PiggyBAC, etc.) and/or RNA transfection.

Also apparent to those of skill in the art is that a variety of methodsof maintaining NK cells under conditions in which (i) all or afunctional portion of the IL-15 is expressed as a membrane-boundpolypeptide and/or as a secreted polypeptide and/or (ii) the NK cellscomprising membrane-bound IL-15 and/or secreted IL-15 proliferate can beused. For example, NK cells can be grown and/or maintained at anappropriate temperature and gas mixture (e.g., about 25° C. to about 37°C., about 5% CO₂ in a cell incubator). Culture conditions can varywidely, and variation of conditions for a particular cell type canresult in different phenotypes. In addition to temperature and gasmixture, a commonly varied factor in culture systems is the cell growthmedium. Recipes for growth media can vary in pH, glucose concentration,growth factors, and the presence of other nutrients. The growth factorsused to supplement media are often derived from the serum of animalblood, such as fetal bovine serum (FBS), bovine calf serum, equineserum, porcine serum and/or human platelet lysate (hPL). Other factorsconsidered for maintaining cells include plating density (number ofcells per volume of culture medium) and growth of the cells insuspension or adherent cultures.

The methods can further comprise isolating or separating the one or moreNK cells produced by the methods provided herein. In addition, themethods can further comprise culturing the one or more NK cells. In someaspects, an NK cell line is produced.

The invention also encompasses a (one or more) natural killer (NK) cellor cell line produced by the methods described herein, and compositionscomprising the NK cells provided herein. In a particular aspect, thecomposition is a pharmaceutical composition comprising one or more ofthe NK cells or cell lines provided herein. The pharmaceuticalcomposition can further comprise all or a functional portion of IL-2(e.g., all or a functional portion of an (one or more) IL-2 protein;nucleic acid encoding all or a functional portion of IL-2).

As used herein, “IL-2” refers to a member of a cytokine family that alsoincludes IL-4, IL-7, IL-9, IL-15 and IL-21. IL-2 signals through areceptor complex consisting of three chains, termed alpha, beta andgamma. The gamma chain is shared by all members of this family ofcytokine receptors. IL-2, which similar to IL-15, facilitates productionof immunoglobulins made by B cells and induces the differentiation andproliferation of NK cells. Primary differences between IL-2 and IL-15are found in adaptive immune responses. For example, IL-2 is necessaryfor adaptive immunity to foreign pathogens, as it is the basis for thedevelopment of immunological memory. On the other hand, IL-15 isnecessary for maintaining highly specific T cell responses by supportingthe survival of CD8 memory T cells.

In another aspect, the invention is directed to a method of treating adisease and/or condition involving NK cell therapy in an individual inneed thereof comprising administering to the individual a natural killer(NK) cell that expresses all or a functional portion of interleukin-15(IL-15). In particular aspects, the NK cells express all or a functionalportion of IL-15 as a membrane-bound polypeptide and/or as a secretedpolypeptide. As is known in the art, diseases and/or conditions thatinvolve NK cell therapy include NK cell deficiencies, cancer, autoimmunediseases, infectious diseases and the like.

In a particular aspect, the invention is directed to a method oftreating cancer (e.g., a tumor) in an individual in need thereofcomprising administering to the individual a natural killer (NK) cellthat expresses all or a functional portion of interleukin-15 (IL-15).All or a functional portion of IL-15 can be expressed as amembrane-bound polypeptide and/or as a secreted polypeptide.

The method can further comprise administering one or more antibodies,antigenic fragments and/or fusions thereof specific to the cancer (e.g.,tumor). For example, the method can further comprise administering oneor more antibodies directed against one or more tumor antigens. As willbe appreciated by those of skill in the art, the one or more antibodiescan be a polyclonal antibody, a monoclonal antibody, a multivalent(e.g., bivalent, trivalent) antibody, a chimeric antibody, a humanizedantibody, etc. and combinations thereof. A variety of antigenicfragments and/or fusions are also known in the art and include Fab′,F(ab′)₂, single chain variable fragment (scFv), multivalent scFv (e.g.,di-scFv, tri-scFv), single domain antibody (nanobody) and etc.

In some aspects, the cancer is a leukemia (e.g., acute lymphoblasticleukemia; acute myeloid leukemia; chronic myelogenous leukemia, chroniclymphocytic leukemia), a myelodysplastic syndrome, a lymphoma (e.g., Bcell non-Hodgkin lymphoma, Hodgkin lymphoma, T-cell lymphoblasticlymphoma, anaplastic large cell lymphoma), a solid tumor (e.g., a breastcancer, prostate cancer, gastric cancer, colon cancer, hepatocellularcarcinoma, nasopharyngeal carcinoma, neuroblastoma, high grade glioma),a sarcoma (e.g., Ewing sarcoma, rhabdomyosarcoma, non-rhabdomyosarcomasoft-tissue sarcoma, osteosarcoma).

The method of treating cancer can further comprise administering IL-2(all or a functional portion of IL-2 protein; nucleic acid encoding allor a functional portion of IL-2) to the individual. In one aspect, theIL-2 is mammalian IL-2, such as human IL-2. In a particular aspect, alow dose of the IL-2 is administered to the individual. As used herein,a “low dose” of IL-12 refers to a dose of IL-2 of about 1 million IU/m²or less (e.g., about 800,000 IU/m²; 600,000 IU/m²; 400,000 IU/m²;200,000 IU/m²; 100,000 IU/m²; 80,000 IU/m²; 60,000 IU/m²; 40,000 IU/m²;20,000 IU/m²; 10,000 IU/m²; 8,000 IU/m²; 6,000 IU/m²; 4,000 IU/m²; 2,000IU/m²; 1,000 IU/m²; 800 IU/m²; 600 IU/m²; 400 IU/m²; 200 IU/m²; 100IU/m²). In contrast, a normal dose of IL-2 is about 1 million IU/m² toabout 5 million IU/m².

The one or more natural killer (NK) cell(s) that express all or afunctional portion of interleukin-15 (IL-15) (e.g., therapeuticcompound; pharmaceutical composition) are administered in atherapeutically effective amount (i.e., an amount that is sufficient totreat the cancer, such as by ameliorating symptoms associated with thecancer, preventing or delaying the onset of the cancer, also lesseningthe severity or frequency of symptoms of the cancer and/or preventing,delaying or overcoming metastasis of the cancer). The amount that willbe therapeutically effective in the treatment of a particular individualwill depend on the symptoms and severity of the condition (e.g.,cancer), and can be determined by standard clinical techniques. Inaddition, in vitro or in vivo assays may optionally be employed to helpidentify optimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the cancer, and should be decided according to thejudgment of a practitioner and each patient's circumstances. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems.

The therapeutic compound can be delivered in a composition (e.g., apharmaceutical composition), as described above, or by themselves. Theycan be administered systemically, or can be targeted to a particulartissue. The therapeutic compounds can be produced by a variety of means,including chemical synthesis; recombinant production; in vivo production(e.g., a transgenic animal, such as U.S. Pat. No. 4,873,316 to Meade etal.), for example, and can be isolated using standard means such asthose described herein. A combination of any of the above methods oftreatment can also be used.

The compounds for use in the methods described herein can be formulatedwith a physiologically acceptable carrier or excipient to prepare apharmaceutical composition. The carrier and composition can be sterile.The formulation should suit the mode of administration.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, dextrose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well ascombinations thereof. The pharmaceutical preparations can, if desired,be mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, flavoring and/or aromatic substances andthe like that do not deleteriously react with the active compounds.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,polyvinyl pyrollidone, sodium saccharine, cellulose, magnesiumcarbonate, etc.

Methods of introduction of these compositions include, but are notlimited to, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, topical, oral and intranasal. Other suitable methods ofintroduction can also include gene therapy (as described below),rechargeable or biodegradable devices, particle acceleration devises(“gene guns”) and slow release polymeric devices. The pharmaceuticalcompositions of this invention can also be administered as part of acombinatorial therapy with other compounds.

The composition can be formulated in accordance with the routineprocedures as a pharmaceutical composition adapted for administration tohuman beings. For example, compositions for intravenous administrationtypically are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampule orsachette indicating the quantity of active compound. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water, salineor dextrose/water. Where the composition is administered by injection,an ampule of sterile water for injection or saline can be provided sothat the ingredients may be mixed prior to administration.

For topical application, nonsprayable forms, viscous to semi-solid orsolid forms comprising a carrier compatible with topical application andhaving a dynamic viscosity preferably greater than water, can beemployed. Suitable formulations include but are not limited tosolutions, suspensions, emulsions, creams, ointments, powders, enemas,lotions, sols, liniments, salves, aerosols, etc., that are, if desired,sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, wetting agents, buffers or salts for influencing osmoticpressure, etc.

Compounds described herein can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In yet other aspects, the invention is directed to pharmaceuticalcompositions comprising one or more NK cells that expresses all or afunctional portion of interleukin-15 (IL-15) as a membrane-boundpolypeptide. The invention is also directed to compositions (e.g.,pharmaceutical compositions) for use as a medicament in therapy. Forexample, the agents identified herein can be used in the treatment ofcancer. In addition, the agents identified herein can be used in themanufacture of a medicament for the treatment of cancer.

As used herein an “individual” refers to an animal, and in a particularaspect, a mammal. Examples of mammals include primates, a canine, afeline, a rodent, and the like. Specific examples include humans, dogs,cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats and mice.The term “individual in need thereof” refers to an individual who is inneed of treatment or prophylaxis as determined by a researcher,veterinarian, medical doctor or other clinician. In one embodiment, anindividual in need thereof is a mammal, such as a human.

An (one or more) “isolated,” “substantially pure,” or “substantiallypure and isolated” NK cell, as used herein, is one that is separatedfrom (substantially isolated with respect to) the complex cellularmilieu in which it naturally occurs, or culture medium when produced byrecombinant techniques, or chemical precursors or other chemicals whenchemically synthesized. In some instances, the isolated material willform part of a composition (for example, a crude extract containingother substances), buffer system, or reagent mix. In othercircumstances, the material may be purified to essential homogeneity,for example, as determined by agarose gel electrophoresis or columnchromatography such as HPLC. Preferably, an NK cell comprises at leastabout 50%, 80%, 90%, 95%, 98% or 99% (on a molar basis) of allmacromolecular species present.

Articles such as “a”, “an”, “the” and the like, may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext.

The phrase “and/or” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined. Multiple elements listed with “and/or” should be construed inthe same fashion, i.e., “one or more” of the elements so conjoined.Other elements may optionally be present other than the elementsspecifically identified by the “and/or” clause. As used herein in thespecification and in the claims, “or” should be understood to have thesame meaning as “and/or” as defined above. For example, when used in alist of elements, “or” or “and/or” shall be interpreted as beinginclusive, i.e., the inclusion of at least one, but optionally more thanone, of list of elements, and, optionally, additional unlisted elements.Only terms clearly indicative to the contrary, such as “only one of” or“exactly one of” will refer to the inclusion of exactly one element of anumber or list of elements. Thus claims that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present, employed in, or otherwiserelevant to a given product or process unless indicated to the contrary.Embodiments are provided in which exactly one member of the group ispresent, employed in, or otherwise relevant to a given product orprocess. Embodiments are provided in which more than one, or all of thegroup members are present, employed in, or otherwise relevant to a givenproduct or process. Any one or more claims may be amended to explicitlyexclude any embodiment, aspect, feature, element, or characteristic, orany combination thereof. Any one or more claims may be amended toexclude any agent, composition, amount, dose, administration route, celltype, target, cellular marker, antigen, targeting moiety, or combinationthereof.

Exemplification

Material and Methods

Tumor Cell Lines

The human cell lines Nalm-6 (B-lineage acute lymphoblastic leukemia),Daudi (B-cell lymphoma), K562 and U937 (acute myeloid leukemia), andSK-BR-3 (breast carcinoma) were obtained from the American Type CultureCollection, the Ewing sarcoma cell line ES8 was from the St. JudeChildren's Research Hospital tissue repository. All of the cell lineswere transduced with a MSCV-internal ribosome entry site (IRES)-GFPretroviral vector (from the St. Jude Vector Development and ProductionShared Resource) containing the firefly luciferase gene. Transducedcells were selected for their expression of GFP with a MoFlo (BeckmanCoulter, Miami, Fla.) or a FACSAria (BD Biosciences, San Jose, Calif.).RPMI-1640 (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetalbovine serum (FBS; Thermo Fisher Scientific, Waltham, Mass.) andantibiotics were used to maintain all cell lines. Cell lines werecharacterized by the providers for molecular and/or gene expressionfeatures; the cell marker profile of leukemia and lymphoma cell lineswas periodically tested by flow cytometry to ensure that no changes hadoccurred and ES8 was validated by DNA fingerprinting at DSMZ(Braunschweig, Germany).

Human NK Cell Expansion

Peripheral blood samples were obtained from discarded byproducts ofplatelet collections from healthy adult donors. Mononuclear cells werepurified by centrifugation on an Accu-Prep density step (Accurate,Westbury, N.Y.) and washed twice in RPMI-1640. To expand CD56+CD3− NKcells, peripheral blood mononuclear cells and the genetically modifiedK562-mb15-41BBL cell line were co-cultured, as previously described inFujisaki et al., Cancer Res, 69(9):4010-4017 (2009); Imai et al., Blood,106:376-383 (2005)). Briefly, peripheral blood mononuclear cells werecultured with 100 Gy-irradiated K562-mb15-41BBL cell at 1.5:1 ratio inSCGM (CellGenix, Freiburg, Germany) containing 10% FBS, antibiotics and10 IU/mL of recombinant human interleukin-2 (IL-2; Roche, Mannheim,Germany) in 6-well tissue culture plates. Tissue culture medium waspartly exchanged every 2 days. After 7 days of co-culture, residual Tcells were removed with Dynabeads CD3 (Invitrogen), resulting in cellpopulation containing >95% CD56+CD3− NK cells.

Plasmids, Virus Production and Gene Transduction

The pMSCV-IRES-GFP, pEQ-PAM3(-E), and pRDF were obtained from the St.Jude Vector Development and Production Shared Resource. Interleukin-15(IL-15) with a long signal peptide was sub-cloned by polymerase chainreaction (PCR) from a human spleen cDNA library (from Dr G. Neale, StJude Children's Research Hospital) used as a template. The cDNA encodingthe signal peptide of CD8α, the mature peptide of IL-15 and thetransmembrane domain of CD8α were assembled by the splicing byoverlapping extension by PCR (SOE-PCR) to encode a membrane-bound formof IL-15 (“mbIL15”); a wild-type form of IL-15 (not linked to CD8αtransmembrane domain; “wtIL15”) was also tested prepared. The resultingexpression cassettes were sub-cloned into EcoRI and XhoI sites of murinestem-cell virus-internal ribosome entry site-green fluorescent protein(MSCV-IRES-GFP).

To generate RD144-pseudotyped retrovirus, 3.0×10⁶ 293 T cells weretransfected using X-tremeGENE 9 DNA (Roche, Mannheim, Germany),maintained in 10-cm tissue culture dishes for 18 h, with 3.5 μg of cDNAencoding mbIL15 constructs, 3.5 μg of pEQ-PAM3(-E), and 3 μg of pRDF.After replacing the medium with RPMI-1640 with 10% FBS and antibioticsat 24 hours, the conditioned medium containing retrovirus was harvestedat 36-96 hours and added to polypropylene tubes coated with RetroNectin(Takara, Otsu, Japan), which were centrifugated at 1400 g for 10 min andincubated at 37° C. and 5% CO₂ for 4 hours. After additionalcentrifugation, and removal of the supernatant, expanded NK cells(0.5-1×10⁶) were added to the tubes and left in at 37° C. for 12 hours;these steps were repeated up to 6 times over 2-3 days. Cells were thenmaintained in RPMI-1640 with FBS, antibiotics and 100 IU/ml of IL-2.Transduced cells were assayed 3-29 days after transduction.

Surface expression of mbIL-15 was analyzed by flow cytometry using ananti-human IL-15 antibody (R&D, Minneapolis, Minn.) and phycoerythrinconjugated goat anti-mouse IgG1 (Southern Biotech, Birmingham, Ala.).Antibody staining was detected with a Fortessa flow cytometer (BectonDickinson). Levels of IL-15 in culture supernatants were measured withthe Quantikine Immunoassay (R&D).

Functional Analysis of NK Cells In Vitro

To estimate NK cell survival and growth in vitro, transduced NK cells(1×10⁶ cells/mL) were resuspended in RPMI-1640 with 10% FBS andantibiotics, placed into the wells of either a 24- or a 96-well plate(Costar, Corning, N.Y.) and cultured without or with IL-2 (10-100IU/ml). Numbers of viable GFP+ cells were determined with an Accuri C6flow cytometer (Becton Dickinson), after staining with propidium iodide.In some experiments, cells were incubated for 10 minutes with aneutralizing anti-IL-15 antibody (R&D) or an isotype-matchednon-reactive antibody before culture.

NK cell immunophenotyping was performed using the antibodies listed inthe Table, visualized with a Fortessa flow cytometer and analyzed byDiva (Becton Dickinson) and FlowJo (TreeStar, Ashland, Oreg.) software.For phosphoprotein analysis, mock- and mbIL15-transduced NK cells(1×10⁷) were cultured without IL-2 for 48 hours. Cell lysates wereprepared using a lysis buffer containing 20 mM 3-(N-morpholino)propanesulfonic acid, 2 mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 60 mMβ-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodiumorthovanadate, 1% Triton X-100, Complete Mini protease inhibitorcocktail (Roche, Mannheim, Germany) and 1 mM dithiothreitol. Aftersonication, lysates were frozen at −80° C. and shipped in dry ice toKinexus (Vancouver, Calif.) for Kinex Antibody Microarray analysis.

For cytotoxicity assays, luciferase-labeled target cells and NK cells(cultured without IL-2 for 48 hours) were plated in 96-well,flat-bottomed black Viewplates (Corning) at various effector:target(E:T) ratios and cultured for 4 or 24 hours. Adherent cell lines wereincubated at 37° C. and 5% CO₂ for 4 hours before adding NK cells toallow for cell attachment. For antibody-dependent cell cytotoxicityassays, Rituximab (Rituxan, Roche; Mannheim, Germany), Trastuzumab(Herceptin, Roche) or purified human IgG (R&D Systems, Minneapolis,Minn.) were added (all at 1 μg/mL) before NK cells. At the end of thecultures, an equal volume of Bright-Glo luciferase reagent (Promega,Madison, Wis.) was then added to each test well, and after 5 minutes,luminescence was measured using a plate reader and analyzed with Gen52.00 software (both from BioTek, Tucson, Ariz.). In each plate, targetcell viability was calculated using the luminescent signal from wellscontaining target cells only. All experiments were done in triplicate.

To measure release of lytic granules, NK cells (cultured for 48 hourswithout IL-2) were cocultured with K562, U937 cells, or 721.221 cellsand their Cw6-expressing variant for 4 hours. We added PE- orPE-Cy7-conjugated anti-CD107a antibody (BD Biosciences) at the beginningof the cultures and GolgiStop (0.15 μL; BD Biosciences) 1 hour later.Percentage of CD107a+NK cells was determined by flow cytometry.

Expansion and Cytotoxicity of NK Cells in Immunodeficient Mice

To test NK cell expansion in vivo, human NK cells transduced with mbIL15or mock-transduced (6-9×106 cells per mouse) were injected in the tailvein of NOD.Cg-Prkdc^(scid) IL2rg^(tm1Wj1)/SzJ (NOD/scid IL2RGnull) mice(Jackson Laboratories, Bar Harbor, Me.). In some mice, we injected 20000IU of IL-2 intraperitoneally (i.p.) 3 times per week. On day 7 and 11,blood cells were counted with a cell counter (Beckman Coulter); humanand mouse CD45+ cells were enumerated by flow cytometry after treatingcells with red cell blood lysis solution (Invitrogen) and staining themwith an allophycocyanin-conjugated mouse-anti-human CD45 and aphycoerythrin-conjugated rat anti-mouse CD45 antibodies (both from BDBiosciences). After euthanasia, human NK cells in bone marrow, liver,spleen, kidney, lung, and brain were enumerated as above. All animalexperiments were performed in accordance a protocol approved by NationalUniversity of Singapore Institutional Animal Care and Use Committee.

To test tumor cell killing in mice, we prepared two xenograft models. Inthe first, U937 cells expressing luciferase were injected i.p. inNOD.Cg-Prkdc^(scid) IL2rg^(tm1Wj1)/SzJ (NOD/scid IL2RGnull) mice (1×10⁴cells per mouse). Three days later, NK cells transduced with the MSCVvector containing either GFP alone or mbIL15 were injected i.p. (1×10⁷cells per mouse); NK cell injection was repeated on day 7. As a control,a group of mice received tissue culture medium instead of NK cells. Inthe second model, mice were engrafted with ES8 cells (i.p.; 1×10⁵ cellsper mouse), followed by 1 NK cell injection on day 3 as above. Tumorengraftment and progression was evaluated using a Xenogen IVIS-200system (Caliper Life Sciences, Hopkinton, Mass.), with imaging beginning5 minutes after i.p. injection of an aqueous solution of D-luciferinpotassium salt (3 mg/mouse). Photons emitted from luciferase-expressioncells were quantified using the Living Image 4.3.1 software program.

Results

Design of IL-15 Constructs and Expression in NK Cells

As described herein, two forms of the IL15 gene were expressed in humanNK cells: a membrane-bound form, resulting from a construct in which thehuman IL15 gene was linked to the gene encoding the transmembrane domainof CD8α (“mbIL15”), and a wild-type unmodified form (“wtIL15”). Bothconstructs were inserted in an MCSV retroviral vector containing GFP(FIG. 1A), which was used to transduce proliferating NK cells obtainedafter culturing peripheral blood mononucleated cells with thestimulatory cell line K562-mb15-41BBL.28 At the end of the cultures,before retroviral transduction, residual T-cells were depleted withanti-CD3 immunomagnetic beads resulting in >95% pure CD56+CD3− cells.Median GFP expression was 71% (23%-97%, n=60) with the constructcontaining mbIL15, and 69% (range, 20%-91%, n=25) with that containingwtIL15. NK cells from the same donors also transduced with a vectorcontaining only GFP had a median GFP expression of 84% (53%-98%, n=60)(FIG. 1B).

After transduction with mbIL15, IL-15 was expressed on the NK cellmembrane: 40%-63% (median, 52; n=7) of GFP+NK cells had IL-15 asdetected by an anti-IL15 antibody (FIG. 1B). By contrast, no IL-15 wasdetectable in cells transduced with wtIL15 (n=4) or mock transduced NKcells (n=7). Production of soluble IL-15 by the transduced NK cells wasdetermined by testing supernatants collected after 24 and 48 hours ofculture. A shown in FIG. 1C, cells expressing wtIL15 secretedsubstantial amounts of IL-15 whereas this was minimal in mbIL15-NK cellsand undetectable in mock-transduced NK cells.

NK Cells Expressing IL-15 have Autonomous Survival and ExpansionCapacity

To determine whether expression of IL-15 could replace exogenous IL-2 inmaintaining NK cell survival, NK cells from 15 donors were transducedwith the mbIL15 construct and cultured in the absence of IL-2; cellnumbers after culture were then compared to those in parallel cultureswith mock-transduced NK cells. As shown in FIG. 2A, expression ofmbIL-15 dramatically increased NK cell survival: after 7 days ofculture, median cell recovery was 85% while virtually no viablemock-transduced NK cell was detectable (<1%; P<0.0001 by paired t test).The effect of mbIL15 significantly decreased if an anti-IL-15neutralizing antibody was added to the cultures (FIGS. 6A-6B). In 9 ofthe 15 donors, recovery of mbIL15 NK cells was also compared to that ofNK cells expressing wtIL15: it was significantly higher with the former(median, 85% versus 56%, P=0.026; FIG. 2A).

In parallel experiments, the supportive effects of IL15 expression inthe presence of exogenous IL-2 were determined. When cultures contained10 IU/mL of IL-2, 7-day recovery of NK cells expressing either mbIL15 orwtIL15 remained significantly higher than that of mock-transduced cells;under these conditions, no significant differences were noted betweenthe 2 forms of IL15 (FIGS. 6A-6B). Only when exogenous IL-2 was presentat a high concentration (100 IU/mL), 7-day recovery of mock-transducedNK cells matched that of NK cells transduced with IL15 (FIG. 6A).

In experiments with expanded NK cells from 6 of the 9 donors, thecapacity of mbIL15 to support NK cell survival beyond 7 days with lowdose IL-2 (10 IU/mL) was determined. On day 14, mbIL15 NK cell numberswere maintained or increased in 4 of the 6 cultures; in 2 of these cellshad further expanded by day 21. Only 2 of the 6 cultures withmock-transduced NK cells from the same donors had maintained cellnumbers on day 14 and 21, and no cell growth was observed; median cellrecovery on day 21 was 205% for mbIL15 NK cells and 80% formock-transduced NK cells. Thus, even in the presence of low dose IL-2,expression mbIL15 conferred a considerable survival and growthadvantage.

In cultures of NK cells from one donor, a particularly high cellrecovery was observed on day 7 when IL15 was expressed (261% with mbIL15and 161% with wtIL15 in the absence of IL-2; 266% and 188% with 10 IU/mLIL-2). These cultures were monitored for 2 months and remarkableimprovements in cell expansion and survival brought about by theexpression of mbIL15 were observed (FIG. 2C). Even in the absence ofIL-2, mblL-15 NK cells continued to survive until day 21 and they werestill detectable 75 days after initiation of the culture, whilemock-transduced cells had become undetectable on day 7 andwtIL15-transduced NK cells on day 42. In the presence of IL-2 at lowconcentration (10 IU/mL), the number of mbIL15-expressing NK cells wasidentical to that originally seeded 2 months after initiation of thecultures, while viable mock-transduced and wtIL15-transduced NK cellshad declined much earlier. As shown in FIG. 6B, when IL-2 was added tothe culture at a high dose (100 IU/mL), NK cells transduced with eithermbIL155 or wtIL15 had a similar persistence profile, both cell typessurviving longer than mock-transduced NK cells even under theseconditions.

Expansion and Homing of mbIL15 NK Cells In Vivo

The experiments performed in vitro indicated that IL15 expressionimproved survival and expansion of NK cells and that mbIL15 producedoverall better stimulation. Whether mbIL15 expression would sustainexpansion of human NK cells in NOD/scid IL2RGnull mice was nextdetermined. Activated NK cells from 4 donors were transduced with mbIL15(52%-74% GFP-positive) and injected into 4 mice (one mouse per donor); 4control mice were injected with mock-transduced NK cells from the samedonors. NK cells expressing mbIL15 expanded much more thanmock-transduced NK cells: 7 days after injection, median number ofmbIL15 NK cells/μ1 of blood was 44.5 (range, 42-60) versus 6.5 (0-12)with mock-transduced NK cells (P=0.004) (FIG. 3A). Parallel experimentswere performed with the same cells, this time also administering 20,000IU human IL-2 i.p. every 2 days (FIG. 3A). Under these conditions,mbIL15 NK cells expanded even more (median NK cells/μl, 101; range,60-167), while mock-transduced cells remained low (median, 18; range,6-20; P=0.021).

On day 11 after injection, mbIL15 NK cells comprised 168.5 cells/μl(range, 94-355) of peripheral blood mononucleated cells in the absenceof IL-2 and 382 cells/μl (151-710) when IL-2 was also administered (FIG.3A, B). By contrast, in mice injected with mock-transduced NK cells,human CD45 cells were virtually undetectable without IL-2, and presentat low levels when IL-2 was also injected (median, 27; range 9-207;P=0.026). Human CD45+ cells also expressed CD56 and lack CD3 (notshown). Of note, the proportion of GFP+ had increased from 66.5%±9.9%before injection to 93.8%±4.4% on day 7 and 94.8%±3.4% on day 11 (P<0.01for both comparisons).

After euthanasia on day 11, 3 of the 4 mice were examined for thepresence of human CD45+ cells in various tissues. If mbIL15 wasexpressed, considerable numbers of human NK cells were detected in bonemarrow, liver, spleen, kidney and lung; in all tissues, numbers weremarkedly higher than those seen with mock-transduced cells (FIG. 3C):mean (±SD) percentage of CD45+ cells expressing mbIL15 was 1.2%±1.5%with no IL-2 and 3.0%±4.3% with IL-2, as compared to 0.04%±0.09% and0.4%±0.6% with mock-transduced cells (P<0.001 and P=0.002,respectively). The only exception was brain where neither mbIL15- normock-transduced NK cells could be detected.

Mechanisms of mbIL15 Stimulation

To determine whether mbIL15 predominantly stimulated cells in trans(IL-15 presented on one NK cell stimulating a neighboring cell (amechanism reported to occur physiologically)) or cis (by direct bindingof mbIL15 to receptors expressed in the same cell), the proportions ofGFP+ and GFP− NK cells in the cultures were evaluated after 7 days ofculture. If the trans mechanism was predominant, the ratio between GFP+and GFP− NK cells should remain unaltered during culture; if cis waspredominant, the proportion of GFP+ cells should increase. FIG. 4A showsthe results of such analysis: the percentage of GFP+ cells among NKcells examined after 7 days of culture without IL-2 consistentlyincreased if mbIL15 was expressed whereas it did not in cultures withmock-transduced cells: GFP+ cells constituted 95.9%±3.3% of the totalcell population versus 57.5%±18.6% on day 7 (P<0.0001), as compared to71.2%±19.0% versus 80.5%±17.1% on day 0. Thus, the predominant mechanismof stimulation by mblL-15 expressed in NK cells is autocrine.

Cells expressing mbIL15 essentially retained the immunophenotype ofactivated NK cells. However, when examined 2 days after IL-2 withdrawalcompared to mock-transduced NK cells, mbIL15 NK cells expressedmoderately higher levels of the activation receptors NKG2D, NKp44(CD336) and NKp30 (CD337) as well as of CD16 and CD56, while expressionof NKp46 (CD335) decreased and that of other molecules, such as DNAM-1(CD226), remained unchanged (FIG. 4B; the Table). The signaltransduction pathways activated by the expression of mbIL15 were alsodetermined. As shown in FIG. 4C, in comparison to mock-transduced NKcells, mblL-15 NK cells had several highly phosphorylated molecules.These included molecules known to be phosphorylated in response to IL-15signaling, such as the transcription factors STAT1, STAT3 and STATS, thekinases src, Erk1/2 and Mek1. Notably, a marked phosphorylation of Bad,as well as phosphorylation of Caspase 7 and 9, collectively indicativeof an anti-apoptotic effect, were observed. Other highly phosphorylatedmolecules in mbIL15 NK cells whose role in IL-15 signaling is unclearincluded CDK6 and RafA.

Effects of mbIL-15 on NK Cell Anti-Tumor Cytotoxicity In Vitro and InVivo

The improvements in NK cell survival and proliferation brought about byexpression of mbIL15 indicated that NK-mediated killing of tumor cellswould likely also increase. This notion was first tested by comparingtumor cell cytotoxicity exerted by mbIL15-NK cells to that of mocktransduced NK cells from the same donors. Experiments with NK cells from9 donors targeting the leukemia cell lines Nalm-6 (B-lineage acutelymphoblastic leukemia), U937 and K562 (acute myeloid leukemia), as wellas Daudi (B-cell lymphoma), SKBR3 (breast carcinoma) and ES8 (Ewingsarcoma) at different E:T ratios and co-culture durations, for a totalof 90 experiments, were performed. FIG. 5A shows results of 24-hourassays: median cytotoxicity was 22% with mock-transduced NK cells at 1:4E:T and 54% at 1:1 E:T; with mbIL15 NK cells, it was 71% and 99%,respectively (P<0.0001). Results with individual cell lines are shown inFIGS. 7A-7B. Although the increased cytotoxicity might be related to theincrease survival of NL cells in culture, an increased release of lyticgranules by mbIL15-NK cells, as revealed by CD107a staining afterculture with either K562 or U937 cells, was also observed (P=0.0067;FIG. 5B).

The gains in vitro cytotoxicity associated with expression of mbIL15were reflected in experiments with NOD/scid IL2RGnull mice engraftedwith human tumor cells. In one set of experiments, mice were injectedwith the human acute myeloid leukemia (AML) cell line U937 and thentreated with either mbIL15- or mock-transduced NK cells. As shown inFIGS. 5C and 5D, mice receiving mbIL15-transduced NK cells had a slowertumor growth and a significantly longer survival than untreated mice andthose treated with mock-transduced NK cells (P=0.014, log rank test fortrend). The cells were also tested in a second xenograft model in whichNOD/scid IL2RGnull mice were injected with the Ewing sarcoma cell lineESB, which has a much slower growth rate, and the mice were treated withone injection of NK cells. As shown in FIGS. 8A-8C, the outcome of micetreated with mbIL15 NK cells (n=12) was superior to that ofmock-transduced NK cells (n=11) and of untreated mice (n=7): mediansurvival was 162, 49 and 21 days, respectively (P=0.005).

DISCUSSION

Among the factors that determine the success of NK cell-based therapy ofcancer, perhaps the most fundamental one is that NK cells persist insufficient numbers to achieve an E:T ratio likely to produce tumorcytoreduction. Demonstrated herein is that expression of amembrane-bound form of IL-15 in human NK cells supported theirautonomous expansion and extended survival in the absence of IL-2. NKcells expressing mbIL15 could be maintained in vitro for up to 2 monthswithout exogenous IL-2. NK cells expressing mbIL15 could expand inimmunodeficient mice and infiltrated multiple tissues where they couldbe found in much larger numbers than mock-transduced cells. Expansion ofmbIL-15 NK cells was further increased by a low concentration of IL-2both in vitro and in vivo. Expression of mbIL15 did not impair thecytotoxic capacity of NK cells. In fact, in xenograft models, mbIL15 NKcells exerted anticancer activity which was more powerful than that ofmock-transduced cells, indicating that this approach might improve theantitumor capacity of NK cell infusions while averting the side effectsof IL-2 administration.

The findings herein show that ectopic expression of IL-15 in human NKcells caused a stronger survival-promoting effect when IL-15 waspresented in a membrane-bound form than in a secreted form. Notably,however, mbIL15 expressed in NK cells preferentially stimulates in cisrather than in the trans when IL-15 is presented by other cells. Thatis, mbIL15 appears to preferentially engage IL-15 receptors on the samecells, resulting in autocrine stimulation. This mechanism explains theIL-15 expression pattern that was consistently observed whenmbIL15-transduced NK cells were labeled with an anti-IL-15 antibody,showing a substantial proportion of cells with strong GFP expression butostensibly lacking IL-15 (FIG. 1B). It is hypothesized that in thesecells IL-15 is expressed but not accessible to antibody because it isbound to its receptor and/or internalized. The capacity of mbIL15 topromote NK cell viability likely explains the increased cytotoxicityexerted by these cells, particularly in 24-hour in vitro assays and invivo. However, the superiority of mbIL15-NK cells was also clear inshort-term (4-hour) assays and these cells also released more lyticgranules according to the CD107a test. Therefore, expression of mbIL15is likely to increase NK cell cytotoxicity by other means, possibly byenhancing their activation status.

Clinical administration of NK cells typically relies on IL-2 to supporttheir survival and expansion in vivo. The multiple side effects relatedto IL-2 administration, however, are potentially serious and oftenrender administration of this cytokine poorly tolerated. Stopping IL-2administration or reducing its dose may results in decreased NK cellexpansion and inefficient anti-tumor effect, which may be furtherinhibited by the stimulation of regulatory T cells. To this end,replacing IL-2 with IL-15 is potentially attractive but the clinicalformulation of IL-15 is still being tested. Although it was overall welltolerated when administered to rhesus macaques, adverse effects wereobserved in some animals, including diarrhea, emesis, weight loss,transient neutropenia, increase in transaminases and hyponatremia. Inaddition to T and NK cell expansion, expansion of regulatory T cells hasbeen observed. Contrary to NK cells transduced with wtIL15, thosetransduced with mbIL15 released exceedingly small amount of IL-15 in thesupernatant. Thus, any potential side effect that may be caused by theinteraction of IL-15 with cells other than NK cells should be minimizedby this approach. Of note, prolonged exposure of murine large granularlymphocytes to IL-15 leads to their leukemic growth. This poses apotential safety concern for IL-15 administration in patients and alsofor the use of NK cells expressing IL-15, particularly if such cellswere administered to patients at a low risk of relapse. In theexperiments described herein, however, NK cells expressing mbIL15generally survived for much shorter periods than the one year or morereported for T cell clones expressing soluble IL-15. Moreover,persistent NK expansion was not observed in immunodeficient mice, with afollow-up exceeding 9 months.

There is considerable clinical evidence supporting the anti-cancerpotential of NK cells. NK cells also play a critical role in mediatingantibody-dependent cell cytotoxicity in patients treated with monoclonalantibodies. Thus, infusion of NK cells is likely beneficial in multiplesettings. Expansion of human NK cells in large numbers ex vivo isfeasible; robust large-scale methods for this purpose have beenestablished and are being used in clinical trials. Genetic modificationof NK cells by retroviral transduction or electroporation is alsopossible. Therefore, the translation of the approach described hereininto clinical-grade conditions is realistic and it is warranted by thesuperior expansion and cytotoxicity of mbIL15-NK cells.

TABLE Surface marker expression in mock- and mbIL15 transduced NK cells¹Mock mb15 Marker %² MFI % MFI CD56 100 44190 100 56721³ CD16 88 7789 9210784  CD69 90 3057 91 4481 CD25 (IL2Rα) 56 631 75  795 CD122(IL2Rβ/IL15Rβ) 100 4833 99 3216 CD132 (IL2Rγ) 89 943 97 1263 NKG2D 992846 100 4953 CD335 (NKp46) 89 2613 89 2236 CD336 (NKp44) 84 9455 8311530  CD337 (NKp30) 91 1286 95 2678 CD226 (DNAM-1) 99 16440 99 16905 CD158ah (KIR2DL1, KIR2DS1 23 6747 24 11793  CD158b 49 42515 47 51247 CD158e 22 4225 22 4946 CD159a 68 18133 73 21106  ¹Cell markers wereanalyzed after 48 hours of culture in the absence of IL-2. Antibodieswere from BD Biosciences (CD56 PE, CD16 PE-Cy7, CD69 PE, CD25 PE-Cy7,CD122 BV421, CD158b PE), Beckman Coulter (CD335 PE, CD336 PE, CD337 PE,CD158ah PE, CD159a PE), Miltenyi Biotech (CD226 PE, CD158e APC), R&DSystems (NKG2D PE), Biolegend (CD132 APC). ²Percentages refer to GFP+cells expressing the marker. ³Overexpressed markers are highlighted inbold font. MFI, mean fluorescence intensity

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-13. (canceled)
 14. A method of treating a cancer, the methodcomprising: administering to a subject having the cancer a population ofcells that express all or a functional portion of interleukin-15(IL-15), wherein the all or a functional portion of the IL-15 is fusedto all or a portion of a transmembrane protein and results in the IL-15being expressed as a cell membrane-bound polypeptide (mbIL15) on thesurface of the cells, wherein the transmembrane protein is selected fromCD8α, CD4, CD3c, CD3γ, CD3δ, CD3, CD28, CD137, glycophorin A,glycophorin D, nicotinic acetylcholine receptor, a GABA receptor,FcεRIγ, or a T-cell receptor, and wherein the all or a functionalportion of the IL-15 comprises all or a functional portion of the aminoacid sequence of SEQ ID NO:
 4. 15. The method of claim 14, wherein thepopulation of cells comprises one or more of natural killer (NK) cells,T-cells, dendritic cells and monocytes.
 16. The method of claim 15,wherein the population of cells comprises isolated CD56⁺CD3⁻ naturalkiller cells.
 17. The method of claim 14, wherein the membrane-boundIL15 activates IL-15 signaling and/or anti-apoptotic signaling in anautocrine manner.
 18. The method of claim 14, wherein the transmembraneprotein comprises CD8α, wherein the mbIL15 comprises the amino acidsequence of SEQ ID NO: 2, and wherein the NK cells expressing all or afunctional portion of IL-15 as a membrane-bound polypeptide exhibitsenhanced expression of one or more activation receptors as compared toNK cells not expressing membrane-bound IL15, wherein the one or moreactivation receptors are selected from NKG2D, NKp44 (CD336) and NKp30(CD337), CD16 or CD56.
 19. The method of claim 14, wherein the cancer isselected from leukemia, a myelodysplastic syndrome, a lymphoma, a solidtumor or a sarcoma.
 20. The method of claim 19, wherein the cancer is aleukemia, and wherein the leukemia is acute lymphoblastic leukemia,acute myeloid leukemia, chronic myelogenous leukemia, or chroniclymphocytic leukemia.
 21. The method of claim 14, further comprisingadministering IL-2 to the individual.
 22. The method of claim 21,wherein the IL-2 is administered at a dose of 1 million IU/m² or less.23. The method of claim 14, further comprising administering one or moreantibodies directed against the cancer to the individual.
 24. A methodof treating a cancer, the method comprising: administering to a subjecthaving the cancer a population of immune cells that expresses all or afunctional portion of interleukin-15 (IL-15), wherein the all or afunctional portion of the IL-15 is fused to all or a portion of atransmembrane protein and results in the IL-15 being expressed as a cellmembrane-bound polypeptide (mbIL15) on the surface of the immune cell,wherein the population of cells comprises one or more of natural killer(NK) cells, T-cells, dendritic cells and monocytes, and wherein the allor a functional portion of IL-15 promotes one or more of: (i) naturalkiller (NK) cell survival, (ii) regulation of NK cell and T cellactivation and proliferation, and (iii) support of NK cell developmentfrom hematopoietic stem cells.
 25. The method of claim 24, wherein theall or a functional portion of the transmembrane protein comprises allor a functional portion of a transmembrane domain of the transmembraneprotein.
 26. The method of claim 24, wherein the transmembrane proteinis selected from CD8α, CD4, CD3ε, CD3γ, CD3δ, CD3ζ, CD28, CD137,glycophorin A, glycophorin D, nicotinic acetylcholine receptor, a GABAreceptor, FcεRIγ, or a T-cell receptor.
 27. The method of claim 24,wherein the all or a functional portion of the IL-15 comprises all or afunctional portion of the amino acid sequence of SEQ ID NO:
 4. 28. Themethod of claim 24, wherein transmembrane protein comprises all or afunctional portion of a CD8α transmembrane protein.
 29. The method ofclaim 24, wherein the mbIL15 comprises the amino acid sequence of SEQ IDNO:
 2. 30. The method of claim 24, wherein the population of cellsexhibits enhanced phosphorylation of one or more of STAT1, STAT3 andSTATS, Src, Erk1/2 or Mek as compared to a population of cells notexpressing membrane-bound IL15, and wherein the population of cellsexpressing all or a functional portion of IL-15 as a membrane-boundpolypeptide exhibits enhanced anti-apoptotic signaling as compared to apopulation of cells not expressing membrane-bound IL15.
 31. A method oftreating a cancer, the method comprising: administering to a subjecthaving the cancer a population of immune cells that expresses all or afunctional portion of interleukin-15 (IL-15), wherein the all or afunctional portion of the IL-15 is fused to all or a portion of a CD8αtransmembrane protein and results in the IL-15 being expressed as a cellmembrane-bound polypeptide (mbIL15) of the NK cell, and wherein the allor a functional portion of IL-15 promotes NK cell survival.
 32. Themethod of claim 31, wherein mbIL15 comprises the amino acid sequence ofSEQ ID NO:
 2. 33. The method of claim 31, further comprisingadministering IL-2 to the individual.
 34. The method of claim 31,wherein the cancer is selected from leukemia, a myelodysplasticsyndrome, a lymphoma, a solid tumor or a sarcoma.